Of the assays which are widely used for determining the presence and quantity of various biological molecules, drugs and the like, radioimmunoassays have been preferred for reasons of sensitivity, accuracy and precision. Because radioimmunoassays employ radioactive isotopes such as iodine-125, many researchers have turned to non-radioactive immunoassays to avoid the need for handling and disposing of radioactive materials and to avoid also the necessity of dealing with a complicated licensing and reporting structure.
Recent technologies have involved the use of immunological assays that employ a reporter molecule that is not radioactive. Of these, luminescent assays are quite comparable to radioimmunoassays in terms of sensitivity, accuracy and precision, and can be extremely fast. One such luminescent assay is described in European Patent Application No. 84300725.3, published Aug. 22, 1984 as Publication No. 116,454 and entitled ENHANCED LUMINESCENT OR LUMINOMETRIC ASSAY, the inventors of which are listed as T. Whitehead, L. Kricka and G. Thorpe. This application describes the use of peroxidase-based conjugates as a tracer system, light being emitted when the peroxidase conjugate is combined with a chemical "cocktail" containing, e.g., luminol as a chemiluminescent reactant, an enhancer compound such as p-iodophenol and an oxidant such as H.sub.2 O.sub.2.
In addition to the European Patent Application described above, reference is made to the following:
Thorpe, G. H. G., Kricka, L. J., Mosely, S. B., and Whitehead, T. B., Phenols As Enhancers Of The Chemiluminescent Horseradish peroxidase-Luminol-Hydrogen Peroxide Reaction: Application in Luminescence-monitored Enzyme Immunoassays, Clin. Chem. 31: 8, 1335-1341 (1985). PA0 Kricka, L. J., and Whitehead T. P. Luminescent Immunoassays: New Labels For An Established Technique. Diagnostic Medicine, May, 1984; pp. 45-52. PA0 Wang, H., George, J., Thorpe, G. H. G., Stott R. A., Kricka, L. J., and Whitehead T. P. Enhanced Luminescence Enzyme Immunoassay For Factor VIII Related Antigen. J. Clin. Pathol. 1985; 38: 317-319. PA0 Thorpe, G. H. G., Williams, L. A., Kricka, L. J., Whitehead, T. P., Evans, H. and Stanworth, D. R. (1985) A Rapid Luminescently Monitored Enzyme Immunoassay For IgE. J. Immuno. Methods 79: 57-63 (1985). PA0 Thorpe, G. H. G., Mose, S. B. Kricka. L. J., Stott, R. A. and Whitehead, T. P. Enhanced Luminescence Determination of Horseradish Peroxidase Conjugates. Anal. Chim. Acta 170: 101-107 (1985). PA0 Whitehead, T. P., Thorpe, G. H. G., Carter, T. J. N., Groucutt, C., and Kricka L. J. Enhanced Luminescence Procedure for Sensitive Determination of Peroxidase-Labelled Conjugates in Immunoassay. Nature 305: 158-159 (1983). PA0 Thorpe, G. H. G., Kricka, L. J., Gillespie, E., Moseley, S., Amess, R., Baggett N. and Whitehead, T. P. Enhancement of the Horseradish Peroxidase- catalyzed Chemiluminescent Oxidation Of Cyclic Diacyl Hydrazides by 6-Hydroxybenzothiazoles. Analytical Biochemistry 145: 96-100 (1985). PA0 Sampson, I., Mattews, J. A., Thorpe, G. H. G., and Kricka, L. J. An Enhanced Luminescence Dot-immunobinding Assay For Cytomegalovirus Antibody Monitored Using Instant Photographic Film. Analytical Letters 18: 1307-1320 (1985). PA0 Thorpe, G. H. G., Haggart, R., Kricka, L. J., and Whitehead, T. P. Enhanced Luminescent Enzyme Immunoassays For Rubella Antibody, Immunoglobulin E and Digoxin. Biochemical and Biophysical Research Communications 119: 481-487 (1984).
A major problem with luminescent assays has been a lack of stability of certain of the luminescent reactants. An additional problem has involved the interference of the luminescent reaction caused by the analyte sample itself. Together, these problems appear to have contributed to the lack of commercialization of immunological tests which employ a luminescent reporter system. We have found that a major drawback with the enhanced luminescent or luminometric assay, referred to above, is the lack of stability of the so-called luminescent "cocktail", a combination of reactants. In addition, we have found that the ability of such cocktails to enter into the light-emitting reaction is severely reduced when the cocktails are maintained at room temperature for more than a few days or are maintained at 37.degree. C. for as short as one day. Thus, such reagent "cocktails" could not withstand elevated temperatures often encountered in normal commercial shipping procedures. Since light is emitted when this cocktail is combined with a peroxidase conjugate, cocktail instability is a serious drawback and limits the usefulness of an otherwise valuable technology.
Additionally, we have found that under certain circumstances, buffer-mediated luminescence, that is, luminescence not caused or "mediated" by peroxidase, can be a significant problem, particularly when high sensitivity is required. Reagents such as the buffers and cocktails described above should desirably have no intrinsic luminescent properties, i.e., they should not emit light other than as a consequence of a peroxidase mediated luminescent reaction.